Gestational diabetes (GDM), diabetes with onset during pregnancy, can result in acute and chronic adverse outcomes for mother and offspring. The overall objective of this proposal is to understand the role of the novel hexokinase gene HKDC1 in the pathogenesis of GDM. In a recent genetic study of the Hyperglycemia and Adverse Pregnancy Outcomes (HAPO) cohort, the PIs and co-authors reported a novel loci associated with maternal glycemic traits specifically during pregnancy. The lead candidate gene in the region, hexokinase- domain containing 1 (HKDC1), has not been characterized. This proposal will test the central hypothesis that HKDC1 is a novel fifth vertebrate hexokinase that controls glucose homeostasis during pregnancy. Testing that hypothesis is important because it will reveal new mechanisms contributing to one of the most common complications of pregnancy worldwide. The results will also have broad-reaching impacts on understanding vertebrate metabolism because hexokinase is the first step in glycolysis, and the dogma in the field is that there are only four vertebrate hexokinase isoforms. Characterizing the function and regulation of the fifth hexokinase will challenge that dogma and will open up several new areas of study. The central hypothesis will be tested at three complementary levels. First, the hexokinase activity of the HKDC1 protein will be extensively characterized. Preliminary data from the PIs already demonstrate that HKDC1 has hexokinase activity. This aim will place HKDC1 in the context of the other hexokinases by systematically characterizing the kinetics of HKDC1 and its subunits. Second, the in vivo role of HKDC1 in glucose homeostasis during pregnancy will be assessed. Tissue expression patterns suggest that HKDC1 regulates glucose homeostasis during pregnancy specifically through its role in pancreatic cells and liver. Through global and tissue-specific ablation mouse models, glucose homeostasis during pregnancy will be assessed. Third, the role of HKDC1 in regulating glucose during human pregnancy will be investigated by testing the hypothesis that GDM-associated genetic variants alter regulation of HKDC1 in the pregnant environment. That hypothesis will be tested using novel high-throughput reporter assays (i) to discover the primary and secondary hormone and glucose response elements that regulate HKDC1 expression and (ii) to identify genetic variants that alter regulatory element function in the original GWAS population. The outcome of this proposal will be that HKDC1 encodes a novel hexokinase whose altered expression impacts glucose homeostasis during pregnancy. This outcome will provide new insight into glucose metabolism during pregnancy.